Bottom Line:
To compare self-complementary (sc) and single-stranded (ss) adeno-associated viral 2/5 (AAV2/5) vectors for retinal cell transduction in the dog when delivered by subretinal injection.These results confirm in a large animal model those previously reported in the mouse.Furthermore, it may be possible to use a lower number of viral particles to achieve the same effect compared with ssAAV2/5 vectors.

Methods: ScAAV2/5 and ssAAV2/5 vectors encoding enhanced green fluorescent protein (GFP) under control of the chicken beta actin promoter were prepared to the same titer. Equal amounts of viral particles were delivered into the subretinal spaces of both eyes of two dogs. In each dog, one eye received the scAAV2/5 and the other the ssAAV2/5. In vivo expression of GFP was monitored ophthalmoscopically. The dogs were sacrificed, and their retinas were examined by fluorescent microscopy and immunohistochemistry to determine GFP expression patterns and to assay for glial reactivity.

Results: GFP expression in the scAAV2/5 injected eyes was detectable at a much earlier time point than in the ssAAV2/5 injected eyes. Expression of GFP was also at higher levels in the scAAV2/5-injected eyes. Expression levels remained stable for the seven month duration of the study. The types of cells transduced by both vectors were similar; there was strong reporter gene expression in the RPE and photoreceptors, although not all cones in the transduced area expressed GFP. Some horizontal and Müller cells were also transduced.

Conclusions: When delivered by subretinal injection in the dog, scAAV2/5 induces faster and stronger transgene expression than ssAAV2/5. The spectrum of retinal neurons transduced is similar between the two vectors. These results confirm in a large animal model those previously reported in the mouse. ScAAV2/5 shows promise for use in the treatment of conditions where a rapid transgene expression is desirable. Furthermore, it may be possible to use a lower number of viral particles to achieve the same effect compared with ssAAV2/5 vectors.

f4: Investigation of GFAP expression in the injected retina. The expression of GFAP was similar between transduced (lower panel) and non-transduced (upper panel) regions of scAAV2/5-injected eyes. The similarity in GFAP immunoreactivity between the injected and non-injected areas indicates that expression of GFP is not inducing glial cell reactivity. Scale bar equals 25 µm.

Mentions:
Retinal pigment epithelium (not shown) and photoreceptors in ssAAV2/5-injected and scAAV2/5-injected eyes expressed GFP (Figure 1B, Figure 2B-F, Figure 3, and Figure 4). Rod cell bodies and inner and outer segments clearly expressed GFP. In both ssAAV2/5- and scAAV2/5-injected eyes some cones in the transduced areas did not express GFP. To further assess cone GFP expression, we used a human cone arrestin (hCAR) antibody that marks the entire cone cell body (Figure 2A-C). This confirmed that within the transduced region, GFP expression was not detectable in all cones (cones not expressing GFP at detectable levels are indicated by arrows in Figure 2D). To assess the proportion of GFP-expressing cones, we counted the number of GFP-positive hCAR immunoreactive cone photoreceptors across sections of the injected area of one of the scAAV2/5 treated retinas. Of 92 consecutive hCAR immunoreactive cones, 55 had clear GFP expression.

f4: Investigation of GFAP expression in the injected retina. The expression of GFAP was similar between transduced (lower panel) and non-transduced (upper panel) regions of scAAV2/5-injected eyes. The similarity in GFAP immunoreactivity between the injected and non-injected areas indicates that expression of GFP is not inducing glial cell reactivity. Scale bar equals 25 µm.

Mentions:
Retinal pigment epithelium (not shown) and photoreceptors in ssAAV2/5-injected and scAAV2/5-injected eyes expressed GFP (Figure 1B, Figure 2B-F, Figure 3, and Figure 4). Rod cell bodies and inner and outer segments clearly expressed GFP. In both ssAAV2/5- and scAAV2/5-injected eyes some cones in the transduced areas did not express GFP. To further assess cone GFP expression, we used a human cone arrestin (hCAR) antibody that marks the entire cone cell body (Figure 2A-C). This confirmed that within the transduced region, GFP expression was not detectable in all cones (cones not expressing GFP at detectable levels are indicated by arrows in Figure 2D). To assess the proportion of GFP-expressing cones, we counted the number of GFP-positive hCAR immunoreactive cone photoreceptors across sections of the injected area of one of the scAAV2/5 treated retinas. Of 92 consecutive hCAR immunoreactive cones, 55 had clear GFP expression.

Bottom Line:
To compare self-complementary (sc) and single-stranded (ss) adeno-associated viral 2/5 (AAV2/5) vectors for retinal cell transduction in the dog when delivered by subretinal injection.These results confirm in a large animal model those previously reported in the mouse.Furthermore, it may be possible to use a lower number of viral particles to achieve the same effect compared with ssAAV2/5 vectors.

Methods: ScAAV2/5 and ssAAV2/5 vectors encoding enhanced green fluorescent protein (GFP) under control of the chicken beta actin promoter were prepared to the same titer. Equal amounts of viral particles were delivered into the subretinal spaces of both eyes of two dogs. In each dog, one eye received the scAAV2/5 and the other the ssAAV2/5. In vivo expression of GFP was monitored ophthalmoscopically. The dogs were sacrificed, and their retinas were examined by fluorescent microscopy and immunohistochemistry to determine GFP expression patterns and to assay for glial reactivity.

Results: GFP expression in the scAAV2/5 injected eyes was detectable at a much earlier time point than in the ssAAV2/5 injected eyes. Expression of GFP was also at higher levels in the scAAV2/5-injected eyes. Expression levels remained stable for the seven month duration of the study. The types of cells transduced by both vectors were similar; there was strong reporter gene expression in the RPE and photoreceptors, although not all cones in the transduced area expressed GFP. Some horizontal and Müller cells were also transduced.

Conclusions: When delivered by subretinal injection in the dog, scAAV2/5 induces faster and stronger transgene expression than ssAAV2/5. The spectrum of retinal neurons transduced is similar between the two vectors. These results confirm in a large animal model those previously reported in the mouse. ScAAV2/5 shows promise for use in the treatment of conditions where a rapid transgene expression is desirable. Furthermore, it may be possible to use a lower number of viral particles to achieve the same effect compared with ssAAV2/5 vectors.